Introduction
Background
Thousand cankers disease (TCD) is caused by the fungal pathogen Geosmithia morbida and its insect vector Pityophthorous juglandis (walnut twig beetle; WTB). The most susceptible TCD host is Juglans nigra (eastern black walnut) an important timber and nut crop native to portions of the eastern and midwestern U.S. TCD has been detected in 15 U.S. states and portions of Italy.
At this time, the effects of G. morbida infection on the J. nigra microbiome are not well understood. Therefore, the objective of this study was to examine the effects of G.moribida infection on the J. nigra phloem microbial communities. We expected that the fungal communities associated with J. nigra phloem tissues would shift in response to G. morbida infection.
Study Design
In the nursery compound at the University of Tennessee Knoxville trees were treated with the following three treatments with 24 trees per treatment:
- Water Control
- Phospho-Jet (Phosphorous Acid-based Fungicide, Arborjet)
- Rootshield (Trichoderma afroharzianum strain T-22, BioWorks)
Of the 72 trees, 36 trees were inoculated with the fungal pathogen Geosmithia morbida isolate TN17 at four separate, equally spaced locations along the stem. This isolate was chosen because it was originally isolated from black walnut in TN and its genome has been sequenced. The remaining 36 trees were inoculated with a potato dextrose agar (PDA) plug to use as an inoculation control. Inoculations took place 7 days following application of Phospho-Jet, Rootshield, and water treatments. On days 14, 28, and 56 post treatment application, trees were destructively sampled with roots and stem tissues sampled for DNA and RNA extraction.
Sample Processing
DNA was extracted from phloem tissues using a CTAB method. Amplified for the ITS2 region using the primers described in Cregger et al. 2018 and sequenced on the Illumina MiSeq platform using 2x250 bp chemistry at the University of Tennessee Genomics Core.
Sequence Processing Methodology
Step 1: Load Packages
Loading in the packages required to complete the sequence processing and statistical analyses.
library(dada2)
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library(Biostrings)
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## get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
## match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
## Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
## table, tapply, union, unique, unsplit, which.max, which.min
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library(ShortRead)
## Loading required package: BiocParallel
## Loading required package: Rsamtools
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library(reticulate)
Step 2: Path Designations
Designating the path for the directory that contains the raw ITS2 sequence data and creating two objects, one for forward paths and the other for reverse paths.
# Creating an object that has the pathway for the sequence directory
phloem_its_path<-"/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing"
# Listing the files present in the sequence directory
list.files(phloem_its_path)
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## [240] "phloem-tree-60-ITS_S27_L001_I1_001.fastq.gz"
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## [242] "phloem-tree-60-ITS_S27_L001_R1_001.fastq.gz"
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## [256] "phloem-tree-64-ITS_S65_L001_I1_001.fastq.gz"
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## [268] "phloem-tree-67-ITS_S45_L001_I1_001.fastq.gz"
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## [271] "phloem-tree-67-ITS_S45_L001_R2_001.fastq.gz"
## [272] "phloem-tree-68-ITS_S53_L001_I1_001.fastq.gz"
## [273] "phloem-tree-68-ITS_S53_L001_I2_001.fastq.gz"
## [274] "phloem-tree-68-ITS_S53_L001_R1_001.fastq.gz"
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## [276] "phloem-tree-69-ITS_S55_L001_I1_001.fastq.gz"
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## [280] "phloem-tree-7-ITS_S13_L001_I1_001.fastq.gz"
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## [283] "phloem-tree-7-ITS_S13_L001_R2_001.fastq.gz"
## [284] "phloem-tree-70-ITS_S64_L001_I1_001.fastq.gz"
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## [290] "phloem-tree-71-ITS_S4_L001_R1_001.fastq.gz"
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## [292] "phloem-tree-72-ITS_S35_L001_I1_001.fastq.gz"
## [293] "phloem-tree-72-ITS_S35_L001_I2_001.fastq.gz"
## [294] "phloem-tree-72-ITS_S35_L001_R1_001.fastq.gz"
## [295] "phloem-tree-72-ITS_S35_L001_R2_001.fastq.gz"
## [296] "phloem-tree-8-ITS_S17_L001_I1_001.fastq.gz"
## [297] "phloem-tree-8-ITS_S17_L001_I2_001.fastq.gz"
## [298] "phloem-tree-8-ITS_S17_L001_R1_001.fastq.gz"
## [299] "phloem-tree-8-ITS_S17_L001_R2_001.fastq.gz"
## [300] "phloem-tree-9-ITS_S11_L001_I1_001.fastq.gz"
## [301] "phloem-tree-9-ITS_S11_L001_I2_001.fastq.gz"
## [302] "phloem-tree-9-ITS_S11_L001_R1_001.fastq.gz"
## [303] "phloem-tree-9-ITS_S11_L001_R2_001.fastq.gz"
# Creating objects for the forward and reverse reads
forwardreads_its_phloem<-sort(list.files(phloem_its_path, pattern="L001_R1_001.fastq",full.names=TRUE))
reversereads_its_phloem<-sort(list.files(phloem_its_path, pattern="L001_R2_001.fastq",full.names=TRUE))
# Checking number of forward and reverse files. Should be an equal number of forward and reverse files.
length(forwardreads_its_phloem)
## [1] 75
length(reversereads_its_phloem)
## [1] 75
# Generating initial forward and reverse read quality plots
initial_forward_qual<-plotQualityProfile(forwardreads_its_phloem, aggregate=TRUE)
initial_forward_qual
initial_reverse_qual<-plotQualityProfile(forwardreads_its_phloem, aggregate=TRUE)
initial_reverse_qual
Step 3: Filtering of Ambiguous Reads
Removing reads with ambiguous bases.
# Creating an object that strips away the pathway information leaving behind only the filename.
filebasename_its_phloem<-basename(forwardreads_its_phloem)
# Cleaning sample names to match with metadata sheet
basename_its_phloem<-sub("-ITS.*","",filebasename_its_phloem)
treebasename_its_phloem<-sub("phloem-","",basename_its_phloem)
numericbasename_its_phloem<-gsub("-","_",treebasename_its_phloem)
# Creating an object that contains only sample names.
samplenames_its_phloem<-numericbasename_its_phloem
# Creating two path objects that will store the paths for filtered forward and reverse reads.
forward.filtN_its_phloem<-file.path(phloem_its_path,"filtN",basename(forwardreads_its_phloem))
reverse.filtN_its_phloem<-file.path(phloem_its_path,"filtN",basename(reversereads_its_phloem))
# Filtering forward and reverse reads using the filterAndTrim function. Specifying maxN=0 will indicate that sequences with at least 1 ambiguous base will be removed from the dataset.
filterAndTrim(forwardreads_its_phloem,forward.filtN_its_phloem,reversereads_its_phloem,reverse.filtN_its_phloem,maxN=0)
# Creating quality profiles for the pre-filtered forward and reverse reads.
filtn_qualplot_fwd_its_phloem<-plotQualityProfile(forward.filtN_its_phloem, aggregate=TRUE)
filtn_qualplot_fwd_its_phloem
filtn_qualplot_rev_its_phloem<-plotQualityProfile(reverse.filtN_its_phloem, aggregate=TRUE)
filtn_qualplot_rev_its_phloem
Step 4: Primer Removal
Removing primers used in the first step of PCR to amplify the ITS2 region. These are low diversity section that will not help with distinguishing between taxa.
# Creating strings that contain the forward and reverse primers used in the study.
its3ngs1<-"CATCGATGAAGAACGCAG"
its3ngs2<-"CAACGATGAAGAACGCAG"
its3ngs3<-"CACCGATGAAGAACGCAG"
its3ngs4<-"CATCGATGAAGAACGTAG"
its3ngs5<-"CATCGATGAAGAACGTGG"
its3ngs10<-"CATCGATGAAGAACGCTG"
its4ngr<-"TCCTSCGCTTATTGATATGC"
archits4<-"TCCTCGCCTTATTGATATGC"
# Creating a function called allorientations to identify all potential orientations of the primers. Then use the complement, reverse, and reverseComplement functions to store all possible orientations of the primer in the orientations object). Last, use the sapply function to convert all orientations into individual strings of text.
allorientations<-function(primer){
require(Biostrings)
dna<-DNAString(primer)
orientations<-c(Forward=dna, Complement=Biostrings::complement(dna),Reverse=Biostrings::reverse(dna),
RevComp=Biostrings::reverseComplement(dna))
return(sapply(orientations,toString))
}
# Storing all possible orientations of primers in objects for each primer.
its3ngs1.ori<-allorientations(its3ngs1)
its3ngs2.ori<-allorientations(its3ngs2)
its3ngs3.ori<-allorientations(its3ngs3)
its3ngs4.ori<-allorientations(its3ngs4)
its3ngs5.ori<-allorientations(its3ngs5)
its3ngs10.ori<-allorientations(its3ngs10)
its4ngr.ori<-allorientations(its4ngr)
archits4.ori<-allorientations(archits4)
# Identifying occurrences of the primers by creating a function called primeroccurences.This function will use the vcountPattern function which will return a vector containing the number of times a particular primer is detected in a sequence and the readFastq function which will take all fastq files in a particular director and turn them into a single object.
primeroccurences<-function(primer, directory) {
nhits<-vcountPattern(primer, sread(readFastq(directory)),fixed=FALSE)
return(sum(nhits>0))
}
# Using the sapply function to apply the primeroccurencesfunction to the directories that contain the forward and reverse reads.The rbind function creates a table for each combination of primer and forward and reverse reads.
primertable_its_phloem<-
rbind(its3ngs1.forwardreads_its_phloem=sapply(its3ngs1.ori,primeroccurences,directory=forward.filtN_its_phloem),
its3ngs1.reversereads_its_phloem=sapply(its3ngs1.ori,primeroccurences,directory=reverse.filtN_its_phloem),
its3ngs2.forwardreads_its_phloem=sapply(its3ngs2.ori,primeroccurences,directory=forward.filtN_its_phloem),
its3ngs2.reversereads_its_phloem=sapply(its3ngs2.ori,primeroccurences,directory=reverse.filtN_its_phloem),
its3ngs3.forwardreads_its_phloem=sapply(its3ngs3.ori,primeroccurences,directory=forward.filtN_its_phloem),
its3ngs3.reversereads_its_phloem=sapply(its3ngs3.ori,primeroccurences,directory=reverse.filtN_its_phloem),
its3ngs4.forwardreads_its_phloem=sapply(its3ngs4.ori,primeroccurences,directory=forward.filtN_its_phloem),
its3ngs4.reversereads_its_phloem=sapply(its3ngs4.ori,primeroccurences,directory=reverse.filtN_its_phloem),
its3ngs5.forwardreads_its_phloem=sapply(its3ngs5.ori,primeroccurences,directory=forward.filtN_its_phloem),
its3ngs5.reversereads_its_phloem=sapply(its3ngs5.ori,primeroccurences,directory=reverse.filtN_its_phloem),
its3ngs10.forwardreads_its_phloem=sapply(its3ngs10.ori,primeroccurences,directory=forward.filtN_its_phloem),
its3ngs10.reversereads_its_phloem=sapply(its3ngs10.ori,primeroccurences,directory=reverse.filtN_its_phloem),
its4ngr.forwardreads_its_phloem=sapply(its4ngr.ori,primeroccurences,directory=forward.filtN_its_phloem),
its4ngr.reversereads_its_phloem=sapply(its4ngr.ori,primeroccurences,directory=reverse.filtN_its_phloem),
archits4.forwardreads_its_phloem=sapply(archits4.ori,primeroccurences,directory=forward.filtN_its_phloem),
archits4.reversereads_its_phloem=sapply(archits4.ori,primeroccurences,directory=reverse.filtN_its_phloem))
primertable_its_phloem
## Forward Complement Reverse RevComp
## its3ngs1.forwardreads_its_phloem 4197059 0 0 2
## its3ngs1.reversereads_its_phloem 241 0 0 335
## its3ngs2.forwardreads_its_phloem 1629310 0 0 0
## its3ngs2.reversereads_its_phloem 72 0 0 166
## its3ngs3.forwardreads_its_phloem 1247251 0 0 2
## its3ngs3.reversereads_its_phloem 107 0 0 140
## its3ngs4.forwardreads_its_phloem 542977 0 0 0
## its3ngs4.reversereads_its_phloem 39 0 0 532
## its3ngs5.forwardreads_its_phloem 10281 0 0 0
## its3ngs5.reversereads_its_phloem 1 0 0 60
## its3ngs10.forwardreads_its_phloem 89814 0 0 0
## its3ngs10.reversereads_its_phloem 9 0 0 31
## its4ngr.forwardreads_its_phloem 264 0 0 789
## its4ngr.reversereads_its_phloem 3631996 0 0 0
## archits4.forwardreads_its_phloem 356 0 0 895
## archits4.reversereads_its_phloem 4130887 0 0 0
# Creating a directory to store the forward and reverse reads after they have been trimmed.
path.cut<-file.path(phloem_its_path,"cutadapt")
if(!dir.exists(path.cut)) dir.create(path.cut)
forwardreads.cut_its_phloem<-file.path(path.cut,basename(forwardreads_its_phloem))
reversereads.cut_its_phloem<-file.path(path.cut,basename(reversereads_its_phloem))
# Creating objects containing the forward and reverse primer reverse compliment strings.The rc function takes a sequence object provided by the user and creates the reverse compliment of the sequence.
its3ngs1.rc1<-dada2::rc(its3ngs1)
its3ngs2.rc1<-dada2::rc(its3ngs2)
its3ngs3.rc1<-dada2::rc(its3ngs3)
its3ngs4.rc1<-dada2::rc(its3ngs4)
its3ngs5.rc1<-dada2::rc(its3ngs5)
its3ngs10.rc1<-dada2::rc(its3ngs10)
its4ngr.rc1<-dada2::rc(its4ngr)
archits4.rc1<-dada2::rc(archits4)
# Using the paste function we then create objects that contain the flags for the potential combinations of each forward and reverse primer and the reverse compliment. These flags will serve as the argumentst that we provide to cutadapt. The ^ at the beginning of the sequence indicates that the primer should be removed from the beginning of the sequence.
its3ngs1.its4.r1.flags<-paste("-a"," ", "^", its3ngs1,"...",its4ngr.rc1, sep='')
its3ngs1.arch.r1.flags<-paste("-a"," ", "^", its3ngs1,"...",archits4.rc1, sep='')
its3ngs2.its4.r1.flags<-paste("-a"," ", "^", its3ngs2,"...",its4ngr.rc1, sep='')
its3ngs2.arch.r1.flags<-paste("-a"," ", "^", its3ngs2,"...",archits4.rc1, sep='')
its3ngs3.its4.r1.flags<-paste("-a"," ", "^", its3ngs3,"...",its4ngr.rc1, sep='')
its3ngs3.arch.r1.flags<-paste("-a"," ", "^", its3ngs3,"...",archits4.rc1, sep='')
its3ngs4.its4.r1.flags<-paste("-a"," ", "^", its3ngs4,"...",its4ngr.rc1, sep='')
its3ngs4.arch.r1.flags<-paste("-a"," ", "^", its3ngs4,"...",archits4.rc1, sep='')
its3ngs5.its4.r1.flags<-paste("-a"," ", "^", its3ngs5,"...",its4ngr.rc1, sep='')
its3ngs5.arch.r1.flags<-paste("-a"," ", "^", its3ngs5,"...",archits4.rc1, sep='')
its3ngs10.its4.r1.flags<-paste("-a"," ", "^", its3ngs10,"...",its4ngr.rc1, sep='')
its3ngs10.arch.r1.flags<-paste("-a"," ", "^", its3ngs10,"...",archits4.rc1, sep='')
its4.its3ngs1.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs1.rc1, sep='')
arch.its3ngs1.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs1.rc1, sep='')
its4.its3ngs2.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs2.rc1, sep='')
arch.its3ngs2.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs2.rc1, sep='')
its4.its3ngs3.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs3.rc1, sep='')
arch.its3ngs3.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs3.rc1, sep='')
its4.its3ngs4.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs4.rc1, sep='')
arch.its3ngs4.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs4.rc1, sep='')
its4.its3ngs5.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs5.rc1, sep='')
arch.its3ngs5.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs5.rc1, sep='')
its4.its3ngs10.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs10.rc1, sep='')
arch.its3ngs10.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs10.rc1, sep='')
# Specifying the conda environment to use for cutadapt
use_condaenv(condaenv="/Users/aarononfurak/anaconda3/envs/cutadaptenv/bin/python")
# Using cutadapt to remove the primers from each read.
for (i in seq_along(forwardreads_its_phloem)){
system2("conda",args=c("run -n cutadaptenv cutadapt",
its3ngs1.its4.r1.flags,
its3ngs1.arch.r1.flags,
its3ngs2.its4.r1.flags,
its3ngs2.arch.r1.flags,
its3ngs3.its4.r1.flags,
its3ngs3.arch.r1.flags,
its3ngs4.its4.r1.flags,
its3ngs4.arch.r1.flags,
its3ngs5.its4.r1.flags,
its3ngs5.arch.r1.flags,
its3ngs10.its4.r1.flags,
its3ngs10.arch.r1.flags,
its4.its3ngs1.r2.flags,
arch.its3ngs1.r2.flags,
its4.its3ngs2.r2.flags,
arch.its3ngs2.r2.flags,
its4.its3ngs3.r2.flags,
arch.its3ngs3.r2.flags,
its4.its3ngs4.r2.flags,
arch.its3ngs4.r2.flags,
its4.its3ngs5.r2.flags,
arch.its3ngs5.r2.flags,
its4.its3ngs10.r2.flags,
arch.its3ngs10.r2.flags,
"--discard-untrimmed","--minimum-length",10,"--report=minimal",
"-o",forwardreads.cut_its_phloem[i], "-p",reversereads.cut_its_phloem[i],
forward.filtN_its_phloem[i],reverse.filtN_its_phloem[i]))
}
# Evaluting the efficacy of primer removal with cutadapt using the primeroccurrences function.
primertable_its_phloem_2<-
rbind(its3ngs1.forwaredreads.cut_its_phloem=sapply(its3ngs1.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
its3ngs1.reversereads.cut_its_phloem=sapply(its3ngs1.ori,primeroccurences,directory=reversereads.cut_its_phloem),
its3ngs2.forwaredreads.cut_its_phloem=sapply(its3ngs2.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
its3ngs2.reversereads.cut_its_phloem=sapply(its3ngs2.ori,primeroccurences,directory=reversereads.cut_its_phloem),
its3ngs3.forwaredreads.cut_its_phloem=sapply(its3ngs3.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
its3ngs3.reversereads.cut_its_phloem=sapply(its3ngs3.ori,primeroccurences,directory=reversereads.cut_its_phloem),
its3ngs4.forwaredreads.cut_its_phloem=sapply(its3ngs4.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
its3ngs4.reversereads.cut_its_phloem=sapply(its3ngs4.ori,primeroccurences,directory=reversereads.cut_its_phloem),
its3ngs5.forwaredreads.cut_its_phloem=sapply(its3ngs5.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
its3ngs5.reversereads.cut_its_phloem=sapply(its3ngs5.ori,primeroccurences,directory=reversereads.cut_its_phloem),
its3ngs10.forwaredreads.cut_its_phloem=sapply(its3ngs10.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
its3ngs10.reversereads.cut_its_phloem=sapply(its3ngs10.ori,primeroccurences,directory=reversereads.cut_its_phloem),
its4ngr.forwaredreads.cut_its_phloem=sapply(its4ngr.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
its4ngr.reversereads.cut_its_phloem=sapply(its4ngr.ori,primeroccurences,directory=reversereads.cut_its_phloem),
archits4.forwaredreads.cut_its_phloem=sapply(archits4.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
archits4.reversereads.cut_its_phloem=sapply(archits4.ori,primeroccurences,directory=reversereads.cut_its_phloem))
primertable_its_phloem_2
## Forward Complement Reverse RevComp
## its3ngs1.forwaredreads.cut_its_phloem 102 0 0 0
## its3ngs1.reversereads.cut_its_phloem 0 0 0 0
## its3ngs2.forwaredreads.cut_its_phloem 11 0 0 0
## its3ngs2.reversereads.cut_its_phloem 0 0 0 0
## its3ngs3.forwaredreads.cut_its_phloem 56 0 0 0
## its3ngs3.reversereads.cut_its_phloem 0 0 0 0
## its3ngs4.forwaredreads.cut_its_phloem 11 0 0 0
## its3ngs4.reversereads.cut_its_phloem 0 0 0 0
## its3ngs5.forwaredreads.cut_its_phloem 0 0 0 0
## its3ngs5.reversereads.cut_its_phloem 0 0 0 0
## its3ngs10.forwaredreads.cut_its_phloem 3 0 0 0
## its3ngs10.reversereads.cut_its_phloem 0 0 0 0
## its4ngr.forwaredreads.cut_its_phloem 0 0 0 0
## its4ngr.reversereads.cut_its_phloem 32 0 0 0
## archits4.forwaredreads.cut_its_phloem 0 0 0 0
## archits4.reversereads.cut_its_phloem 17 0 0 0
# Comparing the pre vs post primer detections.
primertable_its_phloem
## Forward Complement Reverse RevComp
## its3ngs1.forwardreads_its_phloem 4197059 0 0 2
## its3ngs1.reversereads_its_phloem 241 0 0 335
## its3ngs2.forwardreads_its_phloem 1629310 0 0 0
## its3ngs2.reversereads_its_phloem 72 0 0 166
## its3ngs3.forwardreads_its_phloem 1247251 0 0 2
## its3ngs3.reversereads_its_phloem 107 0 0 140
## its3ngs4.forwardreads_its_phloem 542977 0 0 0
## its3ngs4.reversereads_its_phloem 39 0 0 532
## its3ngs5.forwardreads_its_phloem 10281 0 0 0
## its3ngs5.reversereads_its_phloem 1 0 0 60
## its3ngs10.forwardreads_its_phloem 89814 0 0 0
## its3ngs10.reversereads_its_phloem 9 0 0 31
## its4ngr.forwardreads_its_phloem 264 0 0 789
## its4ngr.reversereads_its_phloem 3631996 0 0 0
## archits4.forwardreads_its_phloem 356 0 0 895
## archits4.reversereads_its_phloem 4130887 0 0 0
primertable_its_phloem_2
## Forward Complement Reverse RevComp
## its3ngs1.forwaredreads.cut_its_phloem 102 0 0 0
## its3ngs1.reversereads.cut_its_phloem 0 0 0 0
## its3ngs2.forwaredreads.cut_its_phloem 11 0 0 0
## its3ngs2.reversereads.cut_its_phloem 0 0 0 0
## its3ngs3.forwaredreads.cut_its_phloem 56 0 0 0
## its3ngs3.reversereads.cut_its_phloem 0 0 0 0
## its3ngs4.forwaredreads.cut_its_phloem 11 0 0 0
## its3ngs4.reversereads.cut_its_phloem 0 0 0 0
## its3ngs5.forwaredreads.cut_its_phloem 0 0 0 0
## its3ngs5.reversereads.cut_its_phloem 0 0 0 0
## its3ngs10.forwaredreads.cut_its_phloem 3 0 0 0
## its3ngs10.reversereads.cut_its_phloem 0 0 0 0
## its4ngr.forwaredreads.cut_its_phloem 0 0 0 0
## its4ngr.reversereads.cut_its_phloem 32 0 0 0
## archits4.forwaredreads.cut_its_phloem 0 0 0 0
## archits4.reversereads.cut_its_phloem 17 0 0 0
Step 5: Quality Filtering
Quality filtering data prior to denoising with dada2. Generating quality plots for the sequences that have had their primers removed and use these quality plots to determine what parameters should be used to filter sequences.
# Creating new pathways for the quality filtered forward and reverse reads.
filtforward_its_phloem<-file.path(phloem_its_path, "filtered", paste0(samplenames_its_phloem,"_F_filt.fastq.gz"))
filtreverse_its_phloem<-file.path(phloem_its_path,"filtered",paste0(samplenames_its_phloem,"_R_filt.fastq.gz"))
# Removing paths to non-existent negative control samples that lost all reads following initial quality filtering.
forwardreads.cut_its_phloem_for_qual<-forwardreads.cut_its_phloem[grep("NTC|negative",forwardreads.cut_its_phloem,invert = TRUE)]
reversereads.cut_its_phloem_for_qual<-reversereads.cut_its_phloem[grep("NTC|negative",reversereads.cut_its_phloem,invert = TRUE)]
# Creating quality plot profiles for the primer trimmed forward and reverse reads.
primercut_qualplot_fwd_its_phloem<-plotQualityProfile(forwardreads.cut_its_phloem_for_qual, aggregate=TRUE)
primercut_qualplot_fwd_its_phloem
primercut_qualplot_rev_its_phloem<-plotQualityProfile(reversereads.cut_its_phloem_for_qual, aggregate=TRUE)
primercut_qualplot_rev_its_phloem
# Quality filteirng reads using a maximum expected error threshold of 2 for the forward and reverse reads. Based on the quality plots setting trunlen to 200. This will truncate all reads to 200 bp. The output will be two directories containing filtered forward and filtered reverse reads.
filter.out_its_phloem<-filterAndTrim(fwd=forwardreads.cut_its_phloem, filt=filtforward_its_phloem, rev=reversereads.cut_its_phloem, filt.rev=filtreverse_its_phloem, maxN=0, maxEE=c(2,2), compress=TRUE,truncLen = 200, multithread = FALSE)
## The filter removed all reads: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/1st_AMP_NTC_F_filt.fastq.gz and /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/1st_AMP_NTC_R_filt.fastq.gz not written.
## Warning in file.remove(fout[[1]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/1st_AMP_NTC_F_filt.fastq.gz',
## reason 'No such file or directory'
## Warning in file.remove(fout[[2]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/1st_AMP_NTC_R_filt.fastq.gz',
## reason 'No such file or directory'
## The filter removed all reads: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/2nd_AMP_NTC2_F_filt.fastq.gz and /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/2nd_AMP_NTC2_R_filt.fastq.gz not written.
## Warning in file.remove(fout[[1]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/2nd_AMP_NTC2_F_filt.fastq.gz',
## reason 'No such file or directory'
## Warning in file.remove(fout[[2]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/2nd_AMP_NTC2_R_filt.fastq.gz',
## reason 'No such file or directory'
## The filter removed all reads: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/negative_F_filt.fastq.gz and /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/negative_R_filt.fastq.gz not written.
## Warning in file.remove(fout[[1]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/negative_F_filt.fastq.gz',
## reason 'No such file or directory'
## Warning in file.remove(fout[[2]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/negative_R_filt.fastq.gz',
## reason 'No such file or directory'
## Some input samples had no reads pass the filter.
# The previous step created an object called filter.out_its_phloem. This object is currently a matrix. Converting this object into a dataframe.
filter.out_its_phloem<-as.data.frame(filter.out_its_phloem)
# Determining which sample lost the most number of reads during the filtering process. First creatin a new column that represents the differences between the reads that were fed into the filter and the reads that passed the filter.
filter.out_its_phloem$diffs <- filter.out_its_phloem$reads.in-filter.out_its_phloem$reads.out
# Using the order command to sort the dataframe by the new column (diffs) that was created above.
filter.out_its_phloem[order(filter.out_its_phloem$reads.out),]
## reads.in reads.out diffs
## 1st-AMP-NTC-ITS_S20_L001_R1_001.fastq.gz 0 0 0
## 2nd-AMP-NTC2-ITS_S30_L001_R1_001.fastq.gz 0 0 0
## phloem-negative-ITS_S10_L001_R1_001.fastq.gz 0 0 0
## phloem-tree-11-ITS_S43_L001_R1_001.fastq.gz 27848 9811 18037
## phloem-tree-32-ITS_S24_L001_R1_001.fastq.gz 68178 38330 29848
## phloem-tree-22-ITS_S38_L001_R1_001.fastq.gz 89065 45526 43539
## phloem-tree-5-ITS_S18_L001_R1_001.fastq.gz 79999 49758 30241
## phloem-tree-29-ITS_S70_L001_R1_001.fastq.gz 65046 50941 14105
## phloem-tree-12-ITS_S74_L001_R1_001.fastq.gz 85740 53214 32526
## phloem-tree-4-ITS_S61_L001_R1_001.fastq.gz 78762 53425 25337
## phloem-tree-39-ITS_S58_L001_R1_001.fastq.gz 108120 54538 53582
## phloem-tree-64-ITS_S65_L001_R1_001.fastq.gz 91345 56178 35167
## phloem-tree-1-ITS_S52_L001_R1_001.fastq.gz 87412 58875 28537
## phloem-tree-16-ITS_S68_L001_R1_001.fastq.gz 104051 59413 44638
## phloem-tree-20-ITS_S60_L001_R1_001.fastq.gz 119264 59857 59407
## phloem-tree-2-ITS_S71_L001_R1_001.fastq.gz 125002 60354 64648
## phloem-tree-40-ITS_S32_L001_R1_001.fastq.gz 100625 62315 38310
## phloem-tree-41-ITS_S67_L001_R1_001.fastq.gz 113835 62497 51338
## phloem-tree-35-ITS_S3_L001_R1_001.fastq.gz 131301 62667 68634
## phloem-tree-23-ITS_S49_L001_R1_001.fastq.gz 103122 62733 40389
## phloem-tree-52-ITS_S47_L001_R1_001.fastq.gz 82271 62971 19300
## phloem-tree-31-ITS_S34_L001_R1_001.fastq.gz 92392 63036 29356
## phloem-tree-65-ITS_S69_L001_R1_001.fastq.gz 139614 64781 74833
## phloem-tree-25-ITS_S16_L001_R1_001.fastq.gz 85430 64842 20588
## phloem-tree-50-ITS_S42_L001_R1_001.fastq.gz 131925 65005 66920
## phloem-tree-9-ITS_S11_L001_R1_001.fastq.gz 106159 65019 41140
## phloem-tree-47-ITS_S63_L001_R1_001.fastq.gz 96692 65303 31389
## phloem-tree-61-ITS_S8_L001_R1_001.fastq.gz 100366 66863 33503
## phloem-tree-13-ITS_S2_L001_R1_001.fastq.gz 121481 68451 53030
## phloem-tree-26-ITS_S33_L001_R1_001.fastq.gz 122196 70153 52043
## phloem-tree-48-ITS_S9_L001_R1_001.fastq.gz 106880 70582 36298
## phloem-tree-34-ITS_S36_L001_R1_001.fastq.gz 105150 70922 34228
## phloem-tree-27-ITS_S12_L001_R1_001.fastq.gz 106197 71190 35007
## phloem-tree-18-ITS_S59_L001_R1_001.fastq.gz 119322 71224 48098
## phloem-tree-38-ITS_S37_L001_R1_001.fastq.gz 106100 71593 34507
## phloem-tree-43-ITS_S44_L001_R1_001.fastq.gz 118041 72383 45658
## phloem-tree-54-ITS_S41_L001_R1_001.fastq.gz 110060 72765 37295
## phloem-tree-6-ITS_S19_L001_R1_001.fastq.gz 101923 75019 26904
## phloem-tree-63-ITS_S7_L001_R1_001.fastq.gz 125931 75248 50683
## phloem-tree-7-ITS_S13_L001_R1_001.fastq.gz 125731 75263 50468
## phloem-tree-58-ITS_S25_L001_R1_001.fastq.gz 122170 75790 46380
## phloem-tree-49-ITS_S39_L001_R1_001.fastq.gz 118821 76268 42553
## phloem-tree-46-ITS_S15_L001_R1_001.fastq.gz 109375 76804 32571
## phloem-tree-55-ITS_S57_L001_R1_001.fastq.gz 106983 77083 29900
## phloem-tree-51-ITS_S51_L001_R1_001.fastq.gz 130904 77093 53811
## phloem-tree-70-ITS_S64_L001_R1_001.fastq.gz 113802 77651 36151
## phloem-tree-10-ITS_S56_L001_R1_001.fastq.gz 103002 77878 25124
## phloem-tree-72-ITS_S35_L001_R1_001.fastq.gz 104917 78975 25942
## phloem-tree-42-ITS_S28_L001_R1_001.fastq.gz 122718 79425 43293
## phloem-tree-53-ITS_S22_L001_R1_001.fastq.gz 111978 80129 31849
## phloem-tree-62-ITS_S62_L001_R1_001.fastq.gz 122146 81218 40928
## phloem-tree-57-ITS_S46_L001_R1_001.fastq.gz 98944 81828 17116
## phloem-tree-56-ITS_S66_L001_R1_001.fastq.gz 115947 81980 33967
## phloem-tree-17-ITS_S6_L001_R1_001.fastq.gz 127892 82143 45749
## phloem-tree-21-ITS_S14_L001_R1_001.fastq.gz 114310 82229 32081
## phloem-tree-19-ITS_S73_L001_R1_001.fastq.gz 108635 84202 24433
## phloem-tree-37-ITS_S1_L001_R1_001.fastq.gz 127970 84613 43357
## phloem-tree-28-ITS_S75_L001_R1_001.fastq.gz 119173 84900 34273
## phloem-tree-67-ITS_S45_L001_R1_001.fastq.gz 111190 85673 25517
## phloem-tree-68-ITS_S53_L001_R1_001.fastq.gz 117124 85837 31287
## phloem-tree-14-ITS_S54_L001_R1_001.fastq.gz 129158 86261 42897
## phloem-tree-3-ITS_S23_L001_R1_001.fastq.gz 132941 87889 45052
## phloem-tree-66-ITS_S40_L001_R1_001.fastq.gz 145075 87932 57143
## phloem-tree-69-ITS_S55_L001_R1_001.fastq.gz 123536 90120 33416
## phloem-tree-33-ITS_S50_L001_R1_001.fastq.gz 137983 90353 47630
## phloem-tree-60-ITS_S27_L001_R1_001.fastq.gz 134196 91095 43101
## phloem-tree-24-ITS_S29_L001_R1_001.fastq.gz 127910 93707 34203
## phloem-tree-15-ITS_S72_L001_R1_001.fastq.gz 128910 94590 34320
## phloem-tree-44-ITS_S48_L001_R1_001.fastq.gz 113987 95908 18079
## phloem-tree-45-ITS_S26_L001_R1_001.fastq.gz 122561 98891 23670
## phloem-tree-8-ITS_S17_L001_R1_001.fastq.gz 140443 102315 38128
## phloem-tree-36-ITS_S21_L001_R1_001.fastq.gz 168039 106297 61742
## phloem-tree-30-ITS_S5_L001_R1_001.fastq.gz 143247 110405 32842
## phloem-tree-59-ITS_S31_L001_R1_001.fastq.gz 153485 113092 40393
## phloem-tree-71-ITS_S4_L001_R1_001.fastq.gz 180930 133821 47109
# Removing paths to non-existent negative control samples that lost all reads following initial quality filtering.
filtforward_its_phloem_no_missing<-filtforward_its_phloem[grep("NTC|negative",filtforward_its_phloem,invert = TRUE)]
filtreverse_its_phloem_no_missing<-filtreverse_its_phloem[grep("NTC|negative",filtreverse_its_phloem,invert = TRUE)]
# We then generate quality plots for the forward and reverse reads after quality filtering
filtered_qualplot_fwd_its_phloem<-plotQualityProfile(filtforward_its_phloem_no_missing, aggregate=TRUE)
filtered_qualplot_fwd_its_phloem
filtered_qualplot_rev_its_phloem<-plotQualityProfile(filtreverse_its_phloem_no_missing, aggregate=TRUE)
filtered_qualplot_rev_its_phloem
Step 6: Learn Error Rates
Learning the error rates of the data
# Learning errors for forward and reverse reads.
errorforward_its_phloem<-learnErrors(filtforward_its_phloem_no_missing, multithread = TRUE)
## 101698600 total bases in 508493 reads from 8 samples will be used for learning the error rates.
errorreverse_its_phloem<-learnErrors(filtreverse_its_phloem_no_missing,multithread = TRUE)
## 101698600 total bases in 508493 reads from 8 samples will be used for learning the error rates.
# Error plots for forward & reverse reads
errplots_fwd_its_phloem<-plotErrors(errorforward_its_phloem, nominalQ=TRUE)
errplots_fwd_its_phloem
## Warning: Transformation introduced infinite values in continuous y-axis
## Transformation introduced infinite values in continuous y-axis
errplots_rev_its_phloem<-plotErrors(errorreverse_its_phloem,nominalQ = TRUE)
errplots_rev_its_phloem
## Warning: Transformation introduced infinite values in continuous y-axis
## Transformation introduced infinite values in continuous y-axis
Step 7: Dereplication
Dereplicating reads
# dereplication(merging identical reads into single sequences)
derepforward_its_phloem<-derepFastq(filtforward_its_phloem_no_missing,verbose=TRUE)
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_1_F_filt.fastq.gz
## Encountered 15359 unique sequences from 58875 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_10_F_filt.fastq.gz
## Encountered 11205 unique sequences from 77878 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_11_F_filt.fastq.gz
## Encountered 1355 unique sequences from 9811 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_12_F_filt.fastq.gz
## Encountered 8308 unique sequences from 53214 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_13_F_filt.fastq.gz
## Encountered 7877 unique sequences from 68451 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_14_F_filt.fastq.gz
## Encountered 23277 unique sequences from 86261 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_15_F_filt.fastq.gz
## Encountered 11470 unique sequences from 94590 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_16_F_filt.fastq.gz
## Encountered 9404 unique sequences from 59413 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_17_F_filt.fastq.gz
## Encountered 12082 unique sequences from 82143 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_18_F_filt.fastq.gz
## Encountered 9764 unique sequences from 71224 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_19_F_filt.fastq.gz
## Encountered 10266 unique sequences from 84202 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_2_F_filt.fastq.gz
## Encountered 10407 unique sequences from 60354 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_20_F_filt.fastq.gz
## Encountered 6154 unique sequences from 59857 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_21_F_filt.fastq.gz
## Encountered 13268 unique sequences from 82229 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_22_F_filt.fastq.gz
## Encountered 10086 unique sequences from 45526 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_23_F_filt.fastq.gz
## Encountered 15118 unique sequences from 62733 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_24_F_filt.fastq.gz
## Encountered 9766 unique sequences from 93707 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_25_F_filt.fastq.gz
## Encountered 6347 unique sequences from 64842 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_26_F_filt.fastq.gz
## Encountered 5351 unique sequences from 70153 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_27_F_filt.fastq.gz
## Encountered 8574 unique sequences from 71190 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_28_F_filt.fastq.gz
## Encountered 10134 unique sequences from 84900 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_29_F_filt.fastq.gz
## Encountered 7795 unique sequences from 50941 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_3_F_filt.fastq.gz
## Encountered 6862 unique sequences from 87889 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_30_F_filt.fastq.gz
## Encountered 12327 unique sequences from 110405 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_31_F_filt.fastq.gz
## Encountered 10381 unique sequences from 63036 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_32_F_filt.fastq.gz
## Encountered 7193 unique sequences from 38330 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_33_F_filt.fastq.gz
## Encountered 15310 unique sequences from 90353 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_34_F_filt.fastq.gz
## Encountered 11393 unique sequences from 70922 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_35_F_filt.fastq.gz
## Encountered 6508 unique sequences from 62667 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_36_F_filt.fastq.gz
## Encountered 12617 unique sequences from 106297 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_37_F_filt.fastq.gz
## Encountered 11129 unique sequences from 84613 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_38_F_filt.fastq.gz
## Encountered 11816 unique sequences from 71593 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_39_F_filt.fastq.gz
## Encountered 10191 unique sequences from 54538 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_4_F_filt.fastq.gz
## Encountered 4645 unique sequences from 53425 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_40_F_filt.fastq.gz
## Encountered 8298 unique sequences from 62315 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_41_F_filt.fastq.gz
## Encountered 7260 unique sequences from 62497 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_42_F_filt.fastq.gz
## Encountered 7447 unique sequences from 79425 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_43_F_filt.fastq.gz
## Encountered 7630 unique sequences from 72383 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_44_F_filt.fastq.gz
## Encountered 9419 unique sequences from 95908 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_45_F_filt.fastq.gz
## Encountered 9494 unique sequences from 98891 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_46_F_filt.fastq.gz
## Encountered 17548 unique sequences from 76804 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_47_F_filt.fastq.gz
## Encountered 11489 unique sequences from 65303 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_48_F_filt.fastq.gz
## Encountered 15333 unique sequences from 70582 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_49_F_filt.fastq.gz
## Encountered 11950 unique sequences from 76268 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_5_F_filt.fastq.gz
## Encountered 7909 unique sequences from 49758 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_50_F_filt.fastq.gz
## Encountered 5520 unique sequences from 65005 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_51_F_filt.fastq.gz
## Encountered 4779 unique sequences from 77093 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_52_F_filt.fastq.gz
## Encountered 6598 unique sequences from 62971 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_53_F_filt.fastq.gz
## Encountered 8556 unique sequences from 80129 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_54_F_filt.fastq.gz
## Encountered 15258 unique sequences from 72765 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_55_F_filt.fastq.gz
## Encountered 13325 unique sequences from 77083 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_56_F_filt.fastq.gz
## Encountered 12912 unique sequences from 81980 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_57_F_filt.fastq.gz
## Encountered 10589 unique sequences from 81828 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_58_F_filt.fastq.gz
## Encountered 11434 unique sequences from 75790 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_59_F_filt.fastq.gz
## Encountered 11556 unique sequences from 113092 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_6_F_filt.fastq.gz
## Encountered 12537 unique sequences from 75019 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_60_F_filt.fastq.gz
## Encountered 14445 unique sequences from 91095 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_61_F_filt.fastq.gz
## Encountered 13114 unique sequences from 66863 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_62_F_filt.fastq.gz
## Encountered 18230 unique sequences from 81218 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_63_F_filt.fastq.gz
## Encountered 10116 unique sequences from 75248 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_64_F_filt.fastq.gz
## Encountered 8030 unique sequences from 56178 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_65_F_filt.fastq.gz
## Encountered 9396 unique sequences from 64781 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_66_F_filt.fastq.gz
## Encountered 12037 unique sequences from 87932 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_67_F_filt.fastq.gz
## Encountered 8463 unique sequences from 85673 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_68_F_filt.fastq.gz
## Encountered 12596 unique sequences from 85837 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_69_F_filt.fastq.gz
## Encountered 15204 unique sequences from 90120 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_7_F_filt.fastq.gz
## Encountered 5449 unique sequences from 75263 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_70_F_filt.fastq.gz
## Encountered 19564 unique sequences from 77651 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_71_F_filt.fastq.gz
## Encountered 19252 unique sequences from 133821 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_72_F_filt.fastq.gz
## Encountered 8143 unique sequences from 78975 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_8_F_filt.fastq.gz
## Encountered 16555 unique sequences from 102315 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_9_F_filt.fastq.gz
## Encountered 9881 unique sequences from 65019 total sequences read.
derepreverse_its_phloem<-derepFastq(filtreverse_its_phloem_no_missing,verbose=TRUE)
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_1_R_filt.fastq.gz
## Encountered 11476 unique sequences from 58875 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_10_R_filt.fastq.gz
## Encountered 15459 unique sequences from 77878 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_11_R_filt.fastq.gz
## Encountered 3802 unique sequences from 9811 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_12_R_filt.fastq.gz
## Encountered 12324 unique sequences from 53214 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_13_R_filt.fastq.gz
## Encountered 14817 unique sequences from 68451 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_14_R_filt.fastq.gz
## Encountered 19376 unique sequences from 86261 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_15_R_filt.fastq.gz
## Encountered 18109 unique sequences from 94590 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_16_R_filt.fastq.gz
## Encountered 15731 unique sequences from 59413 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_17_R_filt.fastq.gz
## Encountered 14218 unique sequences from 82143 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_18_R_filt.fastq.gz
## Encountered 18101 unique sequences from 71224 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_19_R_filt.fastq.gz
## Encountered 16875 unique sequences from 84202 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_2_R_filt.fastq.gz
## Encountered 13953 unique sequences from 60354 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_20_R_filt.fastq.gz
## Encountered 18082 unique sequences from 59857 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_21_R_filt.fastq.gz
## Encountered 16072 unique sequences from 82229 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_22_R_filt.fastq.gz
## Encountered 14863 unique sequences from 45526 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_23_R_filt.fastq.gz
## Encountered 11629 unique sequences from 62733 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_24_R_filt.fastq.gz
## Encountered 24409 unique sequences from 93707 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_25_R_filt.fastq.gz
## Encountered 11709 unique sequences from 64842 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_26_R_filt.fastq.gz
## Encountered 16643 unique sequences from 70153 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_27_R_filt.fastq.gz
## Encountered 19714 unique sequences from 71190 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_28_R_filt.fastq.gz
## Encountered 19088 unique sequences from 84900 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_29_R_filt.fastq.gz
## Encountered 10894 unique sequences from 50941 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_3_R_filt.fastq.gz
## Encountered 23131 unique sequences from 87889 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_30_R_filt.fastq.gz
## Encountered 19593 unique sequences from 110405 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_31_R_filt.fastq.gz
## Encountered 11626 unique sequences from 63036 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_32_R_filt.fastq.gz
## Encountered 13465 unique sequences from 38330 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_33_R_filt.fastq.gz
## Encountered 20640 unique sequences from 90353 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_34_R_filt.fastq.gz
## Encountered 15450 unique sequences from 70922 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_35_R_filt.fastq.gz
## Encountered 18851 unique sequences from 62667 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_36_R_filt.fastq.gz
## Encountered 21949 unique sequences from 106297 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_37_R_filt.fastq.gz
## Encountered 18151 unique sequences from 84613 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_38_R_filt.fastq.gz
## Encountered 12350 unique sequences from 71593 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_39_R_filt.fastq.gz
## Encountered 19299 unique sequences from 54538 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_4_R_filt.fastq.gz
## Encountered 15685 unique sequences from 53425 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_40_R_filt.fastq.gz
## Encountered 13763 unique sequences from 62315 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_41_R_filt.fastq.gz
## Encountered 13242 unique sequences from 62497 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_42_R_filt.fastq.gz
## Encountered 23292 unique sequences from 79425 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_43_R_filt.fastq.gz
## Encountered 14923 unique sequences from 72383 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_44_R_filt.fastq.gz
## Encountered 13996 unique sequences from 95908 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_45_R_filt.fastq.gz
## Encountered 14597 unique sequences from 98891 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_46_R_filt.fastq.gz
## Encountered 14533 unique sequences from 76804 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_47_R_filt.fastq.gz
## Encountered 17717 unique sequences from 65303 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_48_R_filt.fastq.gz
## Encountered 12050 unique sequences from 70582 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_49_R_filt.fastq.gz
## Encountered 15890 unique sequences from 76268 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_5_R_filt.fastq.gz
## Encountered 9646 unique sequences from 49758 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_50_R_filt.fastq.gz
## Encountered 16975 unique sequences from 65005 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_51_R_filt.fastq.gz
## Encountered 21977 unique sequences from 77093 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_52_R_filt.fastq.gz
## Encountered 14714 unique sequences from 62971 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_53_R_filt.fastq.gz
## Encountered 21477 unique sequences from 80129 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_54_R_filt.fastq.gz
## Encountered 18268 unique sequences from 72765 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_55_R_filt.fastq.gz
## Encountered 17809 unique sequences from 77083 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_56_R_filt.fastq.gz
## Encountered 16392 unique sequences from 81980 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_57_R_filt.fastq.gz
## Encountered 13930 unique sequences from 81828 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_58_R_filt.fastq.gz
## Encountered 16995 unique sequences from 75790 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_59_R_filt.fastq.gz
## Encountered 23966 unique sequences from 113092 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_6_R_filt.fastq.gz
## Encountered 16922 unique sequences from 75019 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_60_R_filt.fastq.gz
## Encountered 16099 unique sequences from 91095 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_61_R_filt.fastq.gz
## Encountered 12702 unique sequences from 66863 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_62_R_filt.fastq.gz
## Encountered 16768 unique sequences from 81218 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_63_R_filt.fastq.gz
## Encountered 19514 unique sequences from 75248 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_64_R_filt.fastq.gz
## Encountered 16669 unique sequences from 56178 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_65_R_filt.fastq.gz
## Encountered 21161 unique sequences from 64781 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_66_R_filt.fastq.gz
## Encountered 19500 unique sequences from 87932 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_67_R_filt.fastq.gz
## Encountered 18048 unique sequences from 85673 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_68_R_filt.fastq.gz
## Encountered 17471 unique sequences from 85837 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_69_R_filt.fastq.gz
## Encountered 17574 unique sequences from 90120 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_7_R_filt.fastq.gz
## Encountered 23454 unique sequences from 75263 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_70_R_filt.fastq.gz
## Encountered 14791 unique sequences from 77651 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_71_R_filt.fastq.gz
## Encountered 27822 unique sequences from 133821 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_72_R_filt.fastq.gz
## Encountered 20311 unique sequences from 78975 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_8_R_filt.fastq.gz
## Encountered 14875 unique sequences from 102315 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_9_R_filt.fastq.gz
## Encountered 13642 unique sequences from 65019 total sequences read.
# Assigning the sample names to the dereplicated sequence objects
names(derepforward_its_phloem)<-samplenames_its_phloem[grep("AMP|negative",samplenames_its_phloem,invert = TRUE)]
names(derepreverse_its_phloem)<-samplenames_its_phloem[grep("AMP|negative",samplenames_its_phloem,invert = TRUE)]
Step 8: Denoising with DADA2
Now that reads have been quality filtered, error rates have been learned, and reads are dereplicated, proceeding to the sequence inference step of the dada2 pipeline.
#denoise with dada2
dadaforwardreads_its_phloem<-dada(derepforward_its_phloem,err=errorforward_its_phloem,multithread = TRUE,pool=TRUE)
dadareversereads_its_phloem<-dada(derepreverse_its_phloem,err=errorreverse_its_phloem,multithread = TRUE,pool=TRUE)
Step 9: Merge Reads and Chimera Removal
Merging reads and removing chimeric sequences
# First forward and reverse reads are merged using mergePairs.
merge_its_phloem<-mergePairs(dadaforwardreads_its_phloem,filtforward_its_phloem_no_missing,dadareversereads_its_phloem,filtreverse_its_phloem_no_missing,verbose=TRUE)
## 13143 paired-reads (in 404 unique pairings) successfully merged out of 58688 (in 968 pairings) input.
## 74909 paired-reads (in 802 unique pairings) successfully merged out of 77668 (in 1168 pairings) input.
## 9615 paired-reads (in 224 unique pairings) successfully merged out of 9801 (in 279 pairings) input.
## 16728 paired-reads (in 498 unique pairings) successfully merged out of 53061 (in 934 pairings) input.
## 18816 paired-reads (in 429 unique pairings) successfully merged out of 68326 (in 817 pairings) input.
## 40490 paired-reads (in 1039 unique pairings) successfully merged out of 85887 (in 1849 pairings) input.
## 87445 paired-reads (in 726 unique pairings) successfully merged out of 94451 (in 1131 pairings) input.
## 41053 paired-reads (in 403 unique pairings) successfully merged out of 59290 (in 688 pairings) input.
## 14978 paired-reads (in 268 unique pairings) successfully merged out of 82003 (in 626 pairings) input.
## 24391 paired-reads (in 511 unique pairings) successfully merged out of 71091 (in 1022 pairings) input.
## 79106 paired-reads (in 772 unique pairings) successfully merged out of 84001 (in 1123 pairings) input.
## 31118 paired-reads (in 578 unique pairings) successfully merged out of 60202 (in 1046 pairings) input.
## 40030 paired-reads (in 699 unique pairings) successfully merged out of 59708 (in 1116 pairings) input.
## 40836 paired-reads (in 526 unique pairings) successfully merged out of 82010 (in 1042 pairings) input.
## 29358 paired-reads (in 507 unique pairings) successfully merged out of 45279 (in 810 pairings) input.
## 27786 paired-reads (in 413 unique pairings) successfully merged out of 62632 (in 810 pairings) input.
## 73207 paired-reads (in 748 unique pairings) successfully merged out of 93476 (in 1235 pairings) input.
## 60895 paired-reads (in 336 unique pairings) successfully merged out of 64784 (in 504 pairings) input.
## 61741 paired-reads (in 528 unique pairings) successfully merged out of 69975 (in 898 pairings) input.
## 47617 paired-reads (in 730 unique pairings) successfully merged out of 71026 (in 1294 pairings) input.
## 68566 paired-reads (in 654 unique pairings) successfully merged out of 84742 (in 1049 pairings) input.
## 48277 paired-reads (in 630 unique pairings) successfully merged out of 50812 (in 924 pairings) input.
## 83394 paired-reads (in 889 unique pairings) successfully merged out of 87534 (in 1332 pairings) input.
## 94538 paired-reads (in 730 unique pairings) successfully merged out of 110246 (in 1149 pairings) input.
## 14003 paired-reads (in 312 unique pairings) successfully merged out of 62910 (in 652 pairings) input.
## 34180 paired-reads (in 696 unique pairings) successfully merged out of 38096 (in 1078 pairings) input.
## 71506 paired-reads (in 694 unique pairings) successfully merged out of 90139 (in 1129 pairings) input.
## 24293 paired-reads (in 539 unique pairings) successfully merged out of 70668 (in 999 pairings) input.
## 19477 paired-reads (in 674 unique pairings) successfully merged out of 62436 (in 1196 pairings) input.
## 37669 paired-reads (in 698 unique pairings) successfully merged out of 106059 (in 1305 pairings) input.
## 47669 paired-reads (in 754 unique pairings) successfully merged out of 84348 (in 1306 pairings) input.
## 12333 paired-reads (in 411 unique pairings) successfully merged out of 71421 (in 810 pairings) input.
## 37025 paired-reads (in 633 unique pairings) successfully merged out of 54336 (in 1038 pairings) input.
## 29242 paired-reads (in 376 unique pairings) successfully merged out of 53360 (in 630 pairings) input.
## 18124 paired-reads (in 505 unique pairings) successfully merged out of 62123 (in 880 pairings) input.
## 8229 paired-reads (in 253 unique pairings) successfully merged out of 62398 (in 608 pairings) input.
## 61339 paired-reads (in 641 unique pairings) successfully merged out of 79200 (in 997 pairings) input.
## 39759 paired-reads (in 136 unique pairings) successfully merged out of 72266 (in 522 pairings) input.
## 92177 paired-reads (in 611 unique pairings) successfully merged out of 95776 (in 906 pairings) input.
## 96921 paired-reads (in 497 unique pairings) successfully merged out of 98780 (in 724 pairings) input.
## 53502 paired-reads (in 757 unique pairings) successfully merged out of 76617 (in 1324 pairings) input.
## 49606 paired-reads (in 540 unique pairings) successfully merged out of 65185 (in 952 pairings) input.
## 19679 paired-reads (in 404 unique pairings) successfully merged out of 70425 (in 789 pairings) input.
## 13835 paired-reads (in 365 unique pairings) successfully merged out of 75332 (in 858 pairings) input.
## 4985 paired-reads (in 205 unique pairings) successfully merged out of 49603 (in 514 pairings) input.
## 15325 paired-reads (in 343 unique pairings) successfully merged out of 64886 (in 699 pairings) input.
## 67267 paired-reads (in 477 unique pairings) successfully merged out of 77015 (in 750 pairings) input.
## 59787 paired-reads (in 474 unique pairings) successfully merged out of 62842 (in 700 pairings) input.
## 73931 paired-reads (in 770 unique pairings) successfully merged out of 79733 (in 1139 pairings) input.
## 50955 paired-reads (in 678 unique pairings) successfully merged out of 72604 (in 1110 pairings) input.
## 56201 paired-reads (in 663 unique pairings) successfully merged out of 76927 (in 1180 pairings) input.
## 39946 paired-reads (in 768 unique pairings) successfully merged out of 81666 (in 1322 pairings) input.
## 78251 paired-reads (in 623 unique pairings) successfully merged out of 81663 (in 929 pairings) input.
## 23020 paired-reads (in 567 unique pairings) successfully merged out of 75660 (in 992 pairings) input.
## 105158 paired-reads (in 834 unique pairings) successfully merged out of 112858 (in 1309 pairings) input.
## 67937 paired-reads (in 520 unique pairings) successfully merged out of 74547 (in 883 pairings) input.
## 14336 paired-reads (in 442 unique pairings) successfully merged out of 90898 (in 880 pairings) input.
## 29491 paired-reads (in 592 unique pairings) successfully merged out of 66652 (in 1078 pairings) input.
## 21142 paired-reads (in 527 unique pairings) successfully merged out of 80845 (in 1056 pairings) input.
## 24383 paired-reads (in 527 unique pairings) successfully merged out of 75047 (in 1148 pairings) input.
## 34255 paired-reads (in 539 unique pairings) successfully merged out of 56028 (in 1011 pairings) input.
## 49173 paired-reads (in 678 unique pairings) successfully merged out of 64645 (in 1105 pairings) input.
## 34074 paired-reads (in 392 unique pairings) successfully merged out of 87202 (in 856 pairings) input.
## 75397 paired-reads (in 546 unique pairings) successfully merged out of 85580 (in 838 pairings) input.
## 69778 paired-reads (in 554 unique pairings) successfully merged out of 85620 (in 986 pairings) input.
## 66553 paired-reads (in 592 unique pairings) successfully merged out of 89955 (in 979 pairings) input.
## 65254 paired-reads (in 667 unique pairings) successfully merged out of 75095 (in 1070 pairings) input.
## 25511 paired-reads (in 531 unique pairings) successfully merged out of 77474 (in 994 pairings) input.
## 110646 paired-reads (in 1185 unique pairings) successfully merged out of 133423 (in 2016 pairings) input.
## 55318 paired-reads (in 569 unique pairings) successfully merged out of 78773 (in 917 pairings) input.
## 71555 paired-reads (in 573 unique pairings) successfully merged out of 102044 (in 1093 pairings) input.
## 15776 paired-reads (in 424 unique pairings) successfully merged out of 64854 (in 869 pairings) input.
# I then create a sequence table
seqtab_its_phloem<-makeSequenceTable(merge_its_phloem)
hist(nchar(colnames(seqtab_its_phloem)))
# I then remove chimeras using the removeBimeraDenovo function.
seqtab.nochim_its_phloem<-removeBimeraDenovo(seqtab_its_phloem,method="consensus", multithread=TRUE, verbose=TRUE)
## Identified 2480 bimeras out of 4432 input sequences.
#Number of ASVs in the study
ncol((seqtab.nochim_its_phloem))
nrow(seqtab.nochim_its_phloem)
#Number of sequences
sum(seqtab.nochim_its_phloem)
Step 10: Taxonomy Assignment
Assiging taxonomy to sequences from the UNITE database using a modified UNITE database fasta file that includes the ITS2 sequence of Juglans nigra accession MF182372.1.
# Assigning taxonomy using the assignTaxonomy function.
taxa_its_phloem<-assignTaxonomy(seqtab.nochim_its_phloem,"/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sh_general_release_dynamic_25.07.2023_dev.fasta",multithread = TRUE, minBoot=80)
## UNITE fungal taxonomic reference detected.
# Converting the taxonomy assignments to data frame so we can filter the taxonomic assignments.
taxa.original_its_phloem<-as.data.frame(taxa_its_phloem)
# Selecting only ASVs assigned to a fungal kingdom
taxa.no.plant<-taxa.original_its_phloem[grep("Fungi",taxa.original_its_phloem$Kingdom,invert=FALSE),]
# Filtering out ASVs assigned to a fungal phyla
taxa.na.omit_its_phloem<-taxa.no.plant[-(which(is.na(taxa.no.plant$Phylum))),]
Step 11: Taxonomy Merging and ASV Filtering
The last step is to filter the ASV abundance table by merging the ASV table with the taxonomy assignments. This will remove ASVs from the count table that were not assigned to a fungal phyla or were identified as plant sequences.
# Transposing the ASV table so that taxonomy can be added.
t.seqtab.nochim_its_phloem<-t(seqtab.nochim_its_phloem)
# Merging the two tables together based on row name.
t.seqtab.nochim.filt_its_phloem<-t.seqtab.nochim_its_phloem[row.names(t.seqtab.nochim_its_phloem)%in%row.names(taxa.na.omit_its_phloem),]
# Number of ASVs & number of sequences post-filtering
nrow(t.seqtab.nochim.filt_its_phloem)
## [1] 1515
sum(t.seqtab.nochim.filt_its_phloem)
## [1] 2855172
# Merging taxonomy information into ASV table
t.seqtab.tax_its_phloem<-merge(t.seqtab.nochim.filt_its_phloem,taxa.na.omit_its_phloem, by="row.names")
sort(colSums(t.seqtab.nochim.filt_its_phloem))
## tree_5 tree_41 tree_11 tree_38 tree_50 tree_49 tree_1 tree_60 tree_40 tree_9
## 3894 7516 8979 9045 9321 10770 11421 12214 12265 12317
## tree_31 tree_12 tree_17 tree_35 tree_48 tree_13 tree_58 tree_62 tree_34 tree_70
## 12393 12709 13494 14124 16646 16773 18297 18404 19313 20658
## tree_63 tree_18 tree_61 tree_23 tree_2 tree_22 tree_4 tree_66 tree_56 tree_64
## 20964 21614 24159 25006 26653 26798 26945 28608 28646 29015
## tree_36 tree_32 tree_14 tree_37 tree_20 tree_39 tree_27 tree_43 tree_21 tree_29
## 29197 30968 31678 32796 33015 34009 35757 37503 37559 39372
## tree_16 tree_72 tree_46 tree_52 tree_65 tree_54 tree_47 tree_55 tree_42 tree_6
## 39435 41835 43306 43823 44070 45626 46441 51111 53650 54781
## tree_25 tree_26 tree_53 tree_7 tree_3 tree_69 tree_10 tree_8 tree_28 tree_51
## 56451 56603 58128 58468 59725 60107 60832 61745 63494 63832
## tree_24 tree_68 tree_33 tree_67 tree_57 tree_19 tree_15 tree_30 tree_44 tree_59
## 64976 65526 65890 69661 70371 70683 79794 83804 84519 87883
## tree_45 tree_71
## 91249 96538
# Creating ASV labels and make these new row names.
asvnumber_its_phloem<-as.character(c(1:nrow(t.seqtab.nochim.filt_its_phloem)))
asvnumber_its_phloem<-paste("asv_its",labels(asvnumber_its_phloem))
row.names(t.seqtab.tax_its_phloem)<-NULL
row.names(t.seqtab.tax_its_phloem)<-asvnumber_its_phloem
# Pulling out ASV's that are geosmithia
geosmithia.seqs.table<-t.seqtab.tax_its_phloem[grep("Geosmithia",t.seqtab.tax_its_phloem$Genus),]
# Pulling out ASV labels and sequences
geosmithia.seqs<-geosmithia.seqs.table[,1,drop=FALSE]
# Converting the table to a vector object where the ASV names are assigned to the appropriate sequence.
geosmithia.seqs.fmt<-setNames(geosmithia.seqs$Row.names,row.names(geosmithia.seqs))
# Converting to DNA string set for export as a fasta.
geosmithia.seqs.string.set<-Biostrings::DNAStringSet(geosmithia.seqs.fmt)
Biostrings::writeXStringSet(geosmithia.seqs.string.set,"~/Google Drive/My Drive/utk_project/phospho_jet_rootshield_manuscript/manuscript_versions/manuscript_v01/analyses/r_sequence_processing_data/geosmithia.asvs.fa")
write.table(t.seqtab.tax_its_phloem,"~/Google Drive/My Drive/utk_project/phospho_jet_rootshield_manuscript/manuscript_versions/manuscript_v01/analyses/r_sequence_processing_data/feb_22_2024.t.seqtab.tax_its_phloem_manuscript_version.txt",col.names=TRUE,sep='\t',row.names=TRUE)
save.image("~/Google Drive/My Drive/utk_project/phospho_jet_rootshield_manuscript/manuscript_versions/manuscript_v01/analyses/r_sequence_processing_data/feb_22_2024_its_phloem_sequence_processing_manuscript_version.RData")